Fig. 5: Mitochondrial sulfatide regulated BAX localization and directly prevented MOMP. | Cell Death & Differentiation

Fig. 5: Mitochondrial sulfatide regulated BAX localization and directly prevented MOMP.

From: UGT8 mediated sulfatide synthesis modulates BAX localization and dictates apoptosis sensitivity of colorectal cancer

Fig. 5

a CFP-BAX localization was imaged with confocal microscope at 63× magnification. Co01-BAX-KO-CFP-BAX were treated with 2 μM Manidipine-2HCl with or without 50 μM sulfatides for 24 h followed by 5nM A-1155463 stimulation for 3 h. 50 nM Mitotracker Deep Red was used to stain mitochondria; n = 10; b Quantification of the cells positive for CFP-BAX localization to the mitochondria and (c) the average surface areas of the fluorescent spots per cell treated with 2 μM Manidipine-2HCl with or without 50 μM sulfatides for 24 h followed by 5 nM A-1155463; n = 10; d Active BAX immunofluorescent staining with mouse anti-BAX monoclonal antibody 6A7 clone. Co01-BAXKO-CFP-BAX-MitoDsRed were treated with 2 μM Manidipine-2HCl with or without 50 μM sulfatides for 24 h and where indicated followed by 5nM A-1155463 stimulation for 3 h before fixation. Images were obtained with confocal microscope at 63× magnification; e BAX 6A7 staining were analyzed using multiple fields of imaging. Percentage of the cells with 6A7-positive staining was calculated and plotted; n = 6; f Percentage of cell with active BAX(6A7 positive) in those cells with BAX localization (CFP-BAX localization) to mitochondria, n = 6; g MOMP induction was examined by detecting cytosolic cytochrome c (Cyto c) in Co01 cells treated with 2 Manidipine-2HCl and 5nM A-1155463 alone or in combination in the presence or absence of 50 μM sulfatides. Both cytosolic(C) and the rest (containing mitochondrial fraction (M)) of cells were blotted. GAPDH and HSP60 were used as the loading control for cytosolic and mitochondrial fraction respectively. h In vitro cytochrome c release assay was performed on isolated mitochondria within heavy membrane fraction pretreated with or with sulfatides. Both the released fraction (S) and the pellet fraction (P) of mitochondria were blotted. Grayscale of bands was quantified by imageJ and relative release of cytochrome c (supernatant: pellet) was calculated and plotted, n = 3. Significance was calculated with Mann–Whitney test; ns: not significant, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

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